The ASCIZ-DYNLL1 axis promotes 53BP1-dependent non-homologous end joining and PARP inhibitor sensitivity

53BP1 controls a specialized non-homologous end joining (NHEJ) pathway that is essential for adaptive immunity, yet oncogenic in BRCA1 mutant cancers. Intra-chromosomal DNA double-strand break (DSB) joining events during immunoglobulin class switch recombination (CSR) require 53BP1. However, in BRCA1 mutant cells, 53BP1 blocks homologous recombination (HR) and promotes toxic NHEJ, resulting in genomic instability. Here, we identify the protein dimerization hub—DYNLL1—as an organizer of multimeric 53BP1 complexes. DYNLL1 binding stimulates 53BP1 oligomerization, and promotes 53BP1’s recruitment to, and interaction with, DSB-associated chromatin. Consequently, DYNLL1 regulates 53BP1-dependent NHEJ: CSR is compromised upon deletion of Dynll1 or its transcriptional regulator Asciz, or by mutation of DYNLL1 binding motifs in 53BP1; furthermore, Brca1 mutant cells and tumours are rendered resistant to poly-ADP ribose polymerase (PARP) inhibitor treatments upon deletion of Dynll1 or Asciz. Thus, our results reveal a mechanism that regulates 53BP1-dependent NHEJ and the therapeutic response of BRCA1-deficient cancers

The effects of oxidative stress and innate immunity on European badger (Meles meles) life-history dynamics

Wild animals are faced with many stressors from starvation to infections, which will affect individual survival and population dynamics. Free radicals and reactive oxygen species are involved in many of these processes, from cell signalling and immunity, to potentially being a causal factor in ageing. Oxidative stress is posited as a major factor in population dynamics, ultimately driving the evolution of life-history traits in wild species, acting as the 'currency' through which various trade-offs operate. In this thesis, I present a series of studies on the Wytham population of European badgers (Meles meles) focussed on dissecting the effects of host responses to stressors and pathogen recognition on life-history success. The first part of this thesis investigates intrinsic and extrinsic factors affecting oxidative stress and antioxidant defences, linked to population-dynamic effects and life-history traits. I model the crucial intrinsic (development, ageing, reproduction) and one important extrinsic (weather conditions) factors affecting oxidative stress levels in a wild mammal, in relation to survival and reproductive investment. Benefitting from a long term study with marked individuals of a known age, I found that cubs trade-off the development of antioxidant defences against growth, and that juvenile survival is dependent on levels of oxidative damage. These factors are interactive with prevailing yearly weather conditions; investment in antioxidant defences has the greatest survival benefit for young cubs in harsh years (dry, cold spring). I also show that weather affects age-classes in different ways, but I did not find any link between higher oxidative damage and ageing, even following individual responses. The second major focus for the thesis is to explore the immune capability of badgers, focussing on macrophage function and the initial recognition of pathogens. Wildlife immunology represents a "neglected area" of ecology that has potential for large impacts in terms of conservation and disease management. Indeed, wildlife can be a reservoir for infections that affect humans (zoonotic infections) and their livestock. The badger is a classic example of this effect being considered an important reservoir for Mycobacterium bovis, the cause of bovine tuberculosis (bTB). I employed molecular immunology methodologies to develop a methodological tool box to assess immune responses in badgers, which are then employed to investigate the response of blood monocyte-derived macrophages to microbial agonists that stimulate Toll-like receptors. The major findings are that badger macrophages fail to produce nitric oxide or meaningfully upregulate inducible nitric oxide synthase mRNA after exposure to TLR agonists, bacterial lysates and / or recombinant badger Interferon gamma. The TLR system is demonstrated to be largely intact in badger macrophages since exposure to TLR agonists did induce upregulation of cytokine mRNA. The only agonists that stimulated very low responses are those that target TLR9, but this was due to very low levels of expression of TLR9 mRNA. Both nitric oxide and TLR9 are implicated in responses to bTB and these deficiencies would significantly impact on the susceptibility of badgers to infection. There are numerous wider implications of my work: My thesis foremost highlights the importance of taking an integrated approach to eco-physiology and eco-immunology, as these processes are heavily intertwined. The weather correlates of oxidative stress not only highlight the potential vulnerability of all species to human induced rapid environmental change, but provide a stark warning especially for less resilient specialists. The immunology work confirms the importance of carefully analysing immune responses of wildlife species, especially in the context of designing effective species management strategies for diseases such as bTB, providing indicators of why a species may be susceptible to infection and indicating potential ways to improve vaccination.</p

The effects of oxidative stress and innate immunity on European badger (Meles meles) life-history dynamics

Wild animals are faced with many stressors from starvation to infections, which will affect individual survival and population dynamics. Free radicals and reactive oxygen species are involved in many of these processes, from cell signalling and immunity, to potentially being a causal factor in ageing. Oxidative stress is posited as a major factor in population dynamics, ultimately driving the evolution of life-history traits in wild species, acting as the 'currency' through which various trade-offs operate. In this thesis, I present a series of studies on the Wytham population of European badgers (Meles meles) focussed on dissecting the effects of host responses to stressors and pathogen recognition on life-history success. The first part of this thesis investigates intrinsic and extrinsic factors affecting oxidative stress and antioxidant defences, linked to population-dynamic effects and life-history traits. I model the crucial intrinsic (development, ageing, reproduction) and one important extrinsic (weather conditions) factors affecting oxidative stress levels in a wild mammal, in relation to survival and reproductive investment. Benefitting from a long term study with marked individuals of a known age, I found that cubs trade-off the development of antioxidant defences against growth, and that juvenile survival is dependent on levels of oxidative damage. These factors are interactive with prevailing yearly weather conditions; investment in antioxidant defences has the greatest survival benefit for young cubs in harsh years (dry, cold spring). I also show that weather affects age-classes in different ways, but I did not find any link between higher oxidative damage and ageing, even following individual responses. The second major focus for the thesis is to explore the immune capability of badgers, focussing on macrophage function and the initial recognition of pathogens. Wildlife immunology represents a "neglected area" of ecology that has potential for large impacts in terms of conservation and disease management. Indeed, wildlife can be a reservoir for infections that affect humans (zoonotic infections) and their livestock. The badger is a classic example of this effect being considered an important reservoir for Mycobacterium bovis, the cause of bovine tuberculosis (bTB). I employed molecular immunology methodologies to develop a methodological tool box to assess immune responses in badgers, which are then employed to investigate the response of blood monocyte-derived macrophages to microbial agonists that stimulate Toll-like receptors. The major findings are that badger macrophages fail to produce nitric oxide or meaningfully upregulate inducible nitric oxide synthase mRNA after exposure to TLR agonists, bacterial lysates and / or recombinant badger Interferon gamma. The TLR system is demonstrated to be largely intact in badger macrophages since exposure to TLR agonists did induce upregulation of cytokine mRNA. The only agonists that stimulated very low responses are those that target TLR9, but this was due to very low levels of expression of TLR9 mRNA. Both nitric oxide and TLR9 are implicated in responses to bTB and these deficiencies would significantly impact on the susceptibility of badgers to infection. There are numerous wider implications of my work: My thesis foremost highlights the importance of taking an integrated approach to eco-physiology and eco-immunology, as these processes are heavily intertwined. The weather correlates of oxidative stress not only highlight the potential vulnerability of all species to human induced rapid environmental change, but provide a stark warning especially for less resilient specialists. The immunology work confirms the importance of carefully analysing immune responses of wildlife species, especially in the context of designing effective species management strategies for diseases such as bTB, providing indicators of why a species may be susceptible to infection and indicating potential ways to improve vaccination.</p

53BP1 cooperation with the REV7-shieldin complex underpins DNA structure-specific NHEJ.

53BP1 governs a specialized, context-specific branch of the classical non-homologous end joining DNA double-strand break repair pathway. Mice lacking 53bp1 (also known as Trp53bp1) are immunodeficient owing to a complete loss of immunoglobulin class-switch recombination, and reduced fidelity of long-range V(D)J recombination. The 53BP1-dependent pathway is also responsible for pathological joining events at dysfunctional telomeres, and its unrestricted activity in Brca1-deficient cellular and tumour models causes genomic instability and oncogenesis. Cells that lack core non-homologous end joining proteins are profoundly radiosensitive, unlike 53BP1-deficient cells, which suggests that 53BP1 and its co-factors act on specific DNA substrates. Here we show that 53BP1 cooperates with its downstream effector protein REV7 to promote non-homologous end joining during class-switch recombination, but REV7 is not required for 53BP1-dependent V(D)J recombination. We identify shieldin-a four-subunit putative single-stranded DNA-binding complex comprising REV7, c20orf196 (SHLD1), FAM35A (SHLD2) and FLJ26957 (SHLD3)-as the factor that explains this specificity. Shieldin is essential for REV7-dependent DNA end-protection and non-homologous end joining during class-switch recombination, and supports toxic non-homologous end joining in Brca1-deficient cells, yet is dispensable for REV7-dependent interstrand cross-link repair. The 53BP1 pathway therefore comprises distinct double-strand break repair activities within chromatin and single-stranded DNA compartments, which explains both the immunological differences between 53bp1- and Rev7- deficient mice and the context specificity of the pathway